During the past funding period, the PI developed the IVET system for identifying genes of Salmonella typhimurium that are expressed in the animal. The PI's lab will continue to apply this system to Salmonella typhimurium, with the difference that they will alter the system to target secreted proteins. These mutants will be characterized using techniques used to characterize previously obtained mutants. The PI will continue work already begun on genes of H. influenzae that are involved in the aerobic utilization of heme or hemoglobin. Mariner-based transposons developed previously, which allow transposition to be done in vitro, will be used to create new mutants that are affected in their ability to utilize heme. The PI will also continue to work on the virulence factors of V. cholerae. Signature-tag mutagenesis will be applied to V. cholerae and H. influenzae to find genes needed by these organisms to survive in the infant rat model. IVET vectors will also be adapted for use in these two species. The PI has developed a genetic system for Mycobacterium fortuitum, a species that is pathogenic for animals and cultured macrophages. Mutants with lower virulence in these system will be sought using signature tag mutagenesis. Finally, the PI will use the aptamer-based bacterial inhibition system approach to identify inhibitors of regulatory proteins, assembly pathways, and essential genes involved in virulence.